Regulators of mmp-9 and uses therof

ABSTRACT

A method of regulating an activity of metalloproteinase 9 (MMP-9) is disclosed. The method comprises contacting the MMP-9 with an agent which specifically interacts with an OG domain of the MMP-9. Molecules capable of specifically interacting with the OG domain, methods of identifying same, pharmaceutical compositions comprising same and uses thereof are also disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to regulators of MMP-9 and, moreparticularly, to regulators targeted towards the OG domain thereof.

The physiological and pathological roles of matrix metalloproteinases(MMPs) are versatile. Members of the MMP family have been implicated innumerous aspects of the migration of inflammatory and cancer cellsthrough connective tissues, not only by catabolizing extracellularmatrix (ECM) components but also by processing various solublemediators, promoting many disease states. Although all MMPs sharesimilar catalytic sites, marked differences are observed in theirsubstrate specificity, at least in part due to the presence ofadditional substrate binding sites in non-catalytic protein domains. Asa consequence, different MMPs have different biological functions.MMP-9, also known as gelatinase B, is a prototypical target ininflammatory diseases, because of its tissue-damaging roles andinflammation-promoting processing of soluble proteins, includingprotease inhibitors, chemokines and cytokines.

In contrast, MMP-2 or gelatinase A has mainly anti-inflammatory andhomeostatic functions, presumably by the inactivation of inflammatorychemokines and by regulating connective tissue turn-over. This impliesthat selective inhibitors, discriminating between these highly similarenzymes, are crucial for efficient anti-inflammatory therapy withoutside-effects. In this perspective, other non-catalytic parts of theenzyme, differentiating MMP-2 and MMP-9, may be targeted to generateselective inhibitors.

Interestingly, the main structural difference between MMP-9 and MMP-2 isthe presence of an extensively O-glycosylated (OG) domain in MMP-9[Opdenakker, G., et al (2001), Trends Immunol. 22, 571-579; Van denSteen, P. E., et al (2006) J Biol. Chem. 281, 18626-18637]. Otherdomains in MMP-9 are also found in MMP-2 and include a pro-peptidedomain responsible for maintaining latency, a catalytic domain in whichthree fibronectin repeats are inserted, and a C-terminal domain alsoknown as the hemopexin-like domain which constitutes an exosite forbinding of the endogenous MMP-9 and MMP-2 inhibitor, tissue inhibitor ofmetalloproteinase 1 (TIMP-1). Despite its great importance in manydisease states and in contrast to MMP-2, the available structuralinformation about MMP-9 is limited to its two terminal domains, ratherthan the full length enzyme. The X-ray structure of the N-terminal part[Elkins et al, 2002, Acta Crystallogr D Biol Crystallogr 58, 1182-1192],containing the pro-catalytic domain shows that it possesses a matrixinfold. The C-terminal hemopexin-like domain consists of a four-bladedβ-propeller structure with pseudo-four-fold symmetry [Cha et al, 2002, JMol Biol 320, 1065-1079]. FIG. 1A presents the crystal structures of thepro-catalytic and the hemopexin-like domains of pro-MMP-9. The domainsare connected by a dotted line representing the 64 amino acid-longlinker (containing 22 proline residues, 6 glycine residues andapproximately 12-14 O-linked glycans [Van den Steen et al., 2001,Biochim Biophys Acta 1528, 61-73]. Importantly, the linker domain ofpro-MMP-9 is 2-3 times longer than linker regions of collagenases,stromelysins and gelatinase A, of the MMP family, for which typicallinker lengths span a range of only 21-27 amino acid residues.

Crystallization of the linker domain in pro-MMP-9 separately or togetherwith other protein domains has proven difficult. The lack of a largeside chain in the case of glycine and the presence of a built-in bend inthe case of proline interfere with the formation of secondary structureand often result in loops or unstructured regions. In addition, thepresence of clustered serines and threonines as attachment points forO-glycans might yield steric effects that could hinder crystallographicpacking. This domain has also been termed the collagen V-like domain,due to its sequence similarity to collagen V and has recently beenrenamed O-glycosylated (OG) domain. The OG domain is active in theorientation of the hemopexin domains to enable exosite interactions.However, nothing is known of the influence of the OG domain on theoverall 3D structure of MMP-9 and its biophysical nature.

U.S. Patent No. 20040175817 teaches identification of MMP-9 modulatorsbased on the crystal structure of its catalytic subunit. However, sinceMMPs in general share a high sequence homology in their catalytic sites,modulators designed to target the catalytic site will not be selectivetowards MMP-9.

SUMMARY OF THE INVENTION

There is a need for MMP-9 specific regulators.

According to one aspect, there is provided a method of regulating anactivity of metalloproteinase 9 (MMP-9), the method comprisingcontacting the MMP-9 with an agent which specifically interacts with anOG domain of the MMP-9, thereby regulating the activity of MMP-9.

According to another aspect, there is provided a method of identifyingan agent capable of specifically regulating MMP-9, the method comprisingdetermining whether the agent is capable of interacting with an OGdomain of MMP-9, the agent being a putative MMP-9 specific regulator.

According to yet another aspect, there is provided a method of treatinga MMP-9 mediated medical condition, the method comprising administeringto a subject in need thereof a therapeutically effective amount of anagent which specifically interacts with an OG domain of MMP-9, therebytreating the MMP-9 mediated disease or condition.

According to still another aspect, there is provided a molecule capableof specifically regulating an activity of MMP-9, wherein the moleculeinteracts with an OG domain of the MMP-9, with the proviso that themolecule is not a non-humanized antibody.

According to an additional aspect, there is provided a humanizedantibody comprising an antigen recognition domain which specificallyinteract with an OG domain of MMP-9.

According to yet an additional aspect, there is provided apharmaceutical composition comprising as an active ingredient a moleculecapable of specifically regulating an activity of MMP-9, wherein themolecule interacts with an OG domain of the MMP-9, with the proviso thatthe molecule is not a non-humanized antibody and a pharmaceuticallyacceptable carrier.

According to an embodiment, the MMP-9 is native MMP-9.

According to yet another embodiment, the activity is a collagenolyticactivity.

According to yet another embodiment, the activity is a gelatinolyticactivity.

According to yet another embodiment, the regulating is up-regulating.

According to yet another embodiment, the regulating is down-regulating.

According to yet another embodiment, the agent comprises a polypeptideagent.

According to yet another embodiment, the polypeptide agent comprises anantibody.

According to yet another embodiment, the agent comprises a smallmolecule.

According to yet another embodiment, the determining is effected bycomparing a structure of the agent to a structure of an OG domain ofMMP-9.

According to yet another embodiment, the determining is effected bycontacting said agent with an isolated OG domain of MMP-9.

According to yet another embodiment, the agent comprises a polypeptide.

According to yet another embodiment, the polypeptide comprises anantibody.

According to yet another embodiment, the agent comprises a smallmolecule.

According to yet another embodiment, the agent is identified asdescribed herein.

According to yet another embodiment, the agent comprises a smallmolecule or a polypeptide agent.

According to yet another embodiment, the polypeptide agent comprises anantibody.

According to yet another embodiment, the molecule comprises a humanizedantibody comprising an antigen recognition domain which specificallyinteracts with the OG domain of MMP-9.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-D are computer generated models and graphs characterizingpro-MMP-9. FIG. 1A illustrates the crystal structures of the terminaldomains. The N-terminal domain of pro-MMP-9 (PDB code: 1L6J) iscomprised of the pro-peptide (green), three fibronectin type-II repeats(blue) and catalytic domain (red) with the zinc-containing active site(gray sphere). The OG domain (dashed line) contains a 64residue-fragment of unknown structure and it connects the N-terminaldomain to the C-terminal haemopexin-like domain (PDB code: 1ITV), whichconsists four propeller blades (cyan). FIG. 1B is a graph illustratingthe size exclusion chromatography showing the elution profile ofoligomeric species (peak 1, 15.8 min and peak 2, 22.7 min) and monomeric(peak 3, 25.1 min) forms of pro-MMP-9. Inset: Porath plot [57] ofprotein standards with known Stokes radii were used to calibrate thesuperdex 200 column (from left to right: thyroglobulin 85 Å, ferritin 61Å, catalase 52.2 Å, aldolase 48.1 Å, albumin 35.5 Å). The cubic root ofKd is plotted against the Stokes radius of each protein, and linearleast-square fit is shown. FIG. 1C is a photograph of a gelatinzymogram. Glycerol sedimentation was applied in order to separatemonomers from higher oligomeric structures in preparative amounts.Aliquots from each fraction were assayed in a gelatin zymogram. Higholigomeric structures are present in fractions 1-3. Fraction 3 containeda mixture of all oligomeric forms. Fractions 4-7 contained mostly themonomeric form. FIG. 1D is an analytical ultracentrifugationsedimentation velocity analysis, used to calculate the distribution ofsedimentation coefficient. Inset: modeling the sedimentation profiles(lines) from the experimental data (dots) as a function of time anddistance from the axis of rotation. Residuals plot is shown in the upperpanel. For clarity, only every tenth profile used in the analysis isshown.

FIGS. 2A-E are computer generated models and graphs illustrating thestructural analysis of pro-MMP-9. FIG. 2A is a graph illustrating SAXSdata of pro-MMP-9 in solution. Experimental X-ray intensity data (blackdots) are compared with the most probable model (gray line) using CHADD.Inset: pair distribution function of the experimental SAXS data. FIG. 2Billustrates Pro-MMP-9 models reconstructed by CHADD. The models obtainedfrom the SAXS data are represented by white spheres with a radius of 5Å. Each model was rotated at 0° and 90° along the vertical axis. Thedocked crystal structures of the N- and C-terminal domains [22, 24] arerepresented as blue and red ribbons, respectively. FIG. 2C is aprediction of a long-disorder region (thick black line) by PONDR [37] inthe sequence of pro-MMP-9 and the corresponding domain organization (topbar: PRO—pro-peptide, CAT+FN—catalytic domain and three fibronectintype-II repeats, OG—O-glycosylated domain, PEX—hemopexin-like domain).FIG. 2D illustrates the fitting of the calculated scattering curve ofthe full-length pro-MMP-9 with the reconstructed OG domain, to theexperimental data. The calculated curves of the best three models areshown in green, cyan and yellow lines. The experimental data isrepresented as black dots. The three best models of the OG domain werecalculated using RAPPER [38, 39] within the CHADD model. FIG. 2Eillustrates the structural reconstruction of the OG domain. The bestthree models are shown in ribbon representation colored green, cyan andyellow.

FIGS. 3A-F are graphs and AFM images of wild-type and mutated pro-MMP-9.Glutaraldehyde served as covalent linker between the amine on thesurface to the protein. All scans employed a spike tip. FIGS. 3A-C):Semi-dry mode scans of wild-type pro-MMP-9. FIGS. 3D-F: Semi-dry modescan of pro-MMP-9ΔOG mutant. FIG. 3A and FIG. 3D are 2D representation.FIG. 3B and FIG. 3E are 3D representation. FIG. 3C and FIG. 3F are XZcross-sections along the dashed line shown in FIG. 3A and FIG. 3D. Forsample preparation and imaging conditions, see text. Height scale isindicated by the bar to the right in which the Z-axis ranges from 0 to50 Å (dark to light).

FIGS. 4A-F are size distribution histograms of the wild type (left) andpro-MMP-9ΔOG (right) as measured by AFM. The y-axis of all histograms isthe normalized frequency obtained by dividing the counts by the totalpopulation. FIGS. 4A and 4B—height distribution. FIGS. 4C and 4D—widthdistribution. The width values were corrected as described in theexperimental procedures section. FIG. 4E illustrates the lobe-to-lobedistribution of wild type pro-MMP-9. The separation between lobes inpro-MMP-9ΔOG could not be resolved. FIG. 4F illustrates the modelingconformational states of pro-MMP-9. A standard deviation of 9.5 Å, ascalculated according to the lobe-to-lobe AFM data was subtracted (left),or added (right) to the inter-domain separation of the averagedstructure (middle) obtained by SAXS structural reconstruction. The N-and C-terminal domains [22, 24] are represented by blue and red cartoon,respectively. The OG domain was reconstructed by RAPPER [38, 39], and isrepresented by green Cα trace.

FIGS. 5A-B illustrate reconstructed pro-MMP-9 models obtained by SAXS.FIG. 5A is a GASBOR model. FIG. 5B is a CHADD model. White spheres witha radius of 5 Å represent the obtained models. The docked crystalstructures of the N- and C-terminal domains are represented as blue andred cartoons, respectively. Each model was rotated at 0° and 90° alongthe vertical axis.

FIGS. 6A-C are AFM images of pro-MMP-9. Glutaraldehyde served as acovalent linker between the amine on the surface to the protein. Allscans employed a spike tip, except for that of FIG. 6A which used anoxide-sharpened silicon nitride tip. FIG. 6A: Wild type pro-MMP-9 underbuffer solution. FIG. 6B: Desiccated sample of the wild-type enzymescanned in ambient conditions. FIG. 6C: Blank sample subjected to thesame immobilization procedure without applying the enzyme. The arrowindicates a single particle observed on 1×1 μm² scan. Height scale isindicated by the bar to the right in which the Z-axis ranges from 0 to50 Å (dark to light).

FIGS. 7A-B: FIG. 7A illustrates in situ zymography of HT1080 cellsexpressing secreted MMP-9. Detection of green fluorescence is indicativeof proteolytic activity of collagen type IV. Blue indicates nuclearstaining (Hoechst). FIG. 7B illustrates the incubation of HT1080 cellswith Anti-MMP (9 hr) and then overlayed with collagen type-IV conjugatedto Oregon green. Lack of pronounced green fluorescence around the cellsindicates inhibition of pericellular proteolysis by MMP-9.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to regulators of MMP-9 and, moreparticularly, to regulators targeted towards the OG domain thereof.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Members of the metalloproteinase (MMP) family have been implicated innumerous aspects of the migration of inflammatory and cancer cellsthrough connective tissues, promoting many disease states. Although allMMPs share similar catalytic sites, their substrate binding sitesdiffer. As a consequence, different MMPs have different biologicalfunctions. For example, MMP-9 promotes tissue damage and inflammation,whereas MMP-2 comprises mainly anti-inflammatory and homeostaticfunctions. This implies that selective inhibitors, discriminatingbetween these highly similar enzymes, are crucial for efficientanti-inflammatory therapy without side-effects.

Whilst conceiving the invention, the present inventors have come to theunderstanding that the main structural difference between MMP-9 andMMP-2 is the presence of an extensively O-glycosylated (OG) domain inMMP-9. However, the available structural information about MMP-9 islimited to its two terminal domains, which does not include this OGdomain. As such, there is no information pertaining to the influence ofthe OG domain on the overall 3D structure of MMP-9 and its biophysicalnature.

Whilst reducing the present invention to practice, the present inventorshave performed a novel structural analysis combining small-angle X-rayscattering (SAXS) with single molecule atomic force microscopy (AFM)imaging to characterize the first full-length structure of pro-MMP-9 andthe molecular character of its O-glycosylated linker domain. SAXSfollowed by image and structural reconstruction analyses provided themolecular shape of full-length pro-MMP-9 representing its averagedconformation in solution (FIGS. 2A-E). This structure, which issupported by high-resolution AFM imaging (FIGS. 3A-F and 4A-E) andbiophysical measurements, shows an elongated protein with the OG domainacting as a flexible 30 Å long linker between the two terminal domains(FIGS. 5A-B). The degree of the OG domain flexibility was statisticallyevaluated from the various protein conformations detected by singlemolecule imaging (FIG. 4F). The full-length structural-dynamic model ofpro-MMP-9 provides novel insights into the role of protein domainflexibility in the regulation of recognition, binding and processing ofsubstrates, ligands and receptors, required for MMP-9 activities.

Whilst further reducing the present invention to practice, the presentinventors showed by in-situ zymography that an antibody capable ofspecifically interacting with the OG domain of MMP-9, blocks thecollagenolytic activity thereof, but not the gelatinolytic activitythereof (FIGS. 7A-B). Thus, the present inventors suggest that use ofagents that regulate the OG domain flexibility may be used to controlthe pathological activities of this enzyme.

Thus, according to an aspect of the invention, there is provided amethod of regulating an activity of metalloproteinase 9 (MMP-9), themethod comprising contacting the MMP-9 with an agent which specificallyinteracts with an OG domain of the MMP-9, thereby regulating theactivity of MMP-9.

As used herein, the term “MMP-9” (Multidomain zinc endopeptidase matrixmetalloproteinase-9, also named gelatinase B) refers to the precursor oractive forms of the mammalian (e.g., human) MMP-9 polypeptide, (EC3.4.24.35; Swiss Prot No. P14780) including homologs, orthologs andisoforms thereof. MMP-9 typically comprises three domains—a catalyticdomain, a substrate binding domain and a linker domain therebetween. Thelinker domain, also referred to herein as the collagen V-like domain orthe O-glycosylated (OG) domain comprises 64 amino acids, 22 of which areproline residues, 6 of which are glycine residues and approximately12-14 O-linked glycans.

According to an embodiment of this aspect of the invention, the MMP-9 isnative i.e. not denatured. According to another embodiment of thisaspect of the invention, the MMP-9 is active, preferably fully active.

Activities of MMP-9 include, but are not limited to, gelatinolyticactivities, degradation of native collagens of type I, III and XI(collagenolytic activities), degradation of elastin, aggrecan, thelaminin A chain and myelin basic protein.

The term “regulating” as used herein refers to down-regulating orup-regulating. It will be appreciated that agents which inhibit theflexibility of the OG domain will down-regulate a function of MMP-9 thatrequire the OG domain to be flexible, such as its collagenolyticactivity. In contrast, activities which require a particular 3Dstructure of MMP-9 and do not require flexibility of the OG domain maybe up-regulated by agents interacting with the OG domain. An example ofsuch an activity is its gelatinolytic activity or an ability to interactwith receptors and/or growth factors.

As mentioned, the method of the invention is effected by contactingMMP-9 with an agent capable of specifically interacting with its OGdomain.

As used herein, the term “contacting” refers to enabling MMP-9 to comeinto contact with the agent under conditions (i.e. time, temperature,buffer) that allow the agent to interact with its OG domain (e.g. bindto the OG domain) and affect rigidity thereof. It will be appreciatedthat the contacting may be effected in vivo, ex vivo or in vitro.

The phrase “specifically interacting”, as used herein refers to both anenhanced affinity towards the OG domain of MMP-9 as opposed to anotherdomain of MMP-9 (e.g. catalytic domain or substrate binding domain) andan enhanced affinity towards the OG domain of MMP-9 over an OG domain ofanother metalloproteinase enzyme e.g. MMP-2. An example of minimalaffinity is probably 10⁻⁵M. Preferably the agent interacts with the MP-9OG domain with at least 3 times higher affinity as compared to theabove, more preferably, with at least 5 times higher affinity, morepreferably with at least 10 times higher affinity or greater. It will beappreciated that since the amino acid sequence of the MMP-9 OG domain isspecific to MMP-9 (as opposed to the amino acid sequence of thecatalytic domain which is highly homologous between MMP-9 and MMP-2),agents capable of specifically interacting with the MMP-9 OG domain arethus capable of specifically regulating MMP-9.

Agents (i.e. molecules) contemplated by the present invention capable ofinteracting with the OG domain of MMP-9 include, but are not limited topolypeptide agents (e.g. antibodies comprising an antigen recognitiondomain which specifically interact with an OG domain of MMP-9), peptidesand small molecules. It will be appreciated that the agents may interactwith the OG domain based on specific amino acid sequence recognitionand/or conformational recognition.

Antibody agents that recognize the OG domain of MMP-9 are commerciallyavailable, e.g., from Sigma, Chemicon and Abcam.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to the specific mitochondrial proteins.Smaller antibody fragments may be advantageous over whole antibodiessince they are able to penetrate tissue more readily and are morerapidly cleared from the body. This is especially relevant for thein-vivo use of MMP-9 specific antibodies. Also, an additional advantageof antibody fragments is that they may be produced in bacteria oryeasts.

Generation of antibodies directed against the OG domain of MMP-9 may beeffected by using a peptide which comprises the OG domain. The antibodymay be selected using other MMP-9 domains as negative controls.

Suitable Antibody fragments for practicing the present invention includea complementarity-determining region (CDR) of an immunoglobulin lightchain (referred to herein as “light chain”), acomplementarity-determining region of an immunoglobulin heavy chain(referred to herein as “heavy chain”), a variable region of a lightchain, a variable region of a heavy chain, a light chain, a heavy chain,an Fd fragment, and antibody fragments comprising essentially wholevariable regions of both light and heavy chains such as an Fv, a singlechain Fv, an Fab, an Fab′, and an F(ab′)2.

Functional antibody fragments comprising whole or essentially wholevariable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of thevariable region of the light chain and the variable region of the heavychain expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker.

(iii) Fab, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme papain to yield the intact lightchain and the Fd fragment of the heavy chain which consists of thevariable and CH1 domains thereof;

(iv) Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin, followed by reduction(two Fab′ fragments are obtained per antibody molecule); and

(v) F(ab′)2, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin (i.e., a dimer of Fab′fragments held together by two disulfide bonds).

Methods of generating antibodies (i.e., monoclonal and polyclonal) arewell known in the art. Antibodies may be generated via any one ofseveral methods known in the art, which methods can employ induction ofin-vivo production of antibody molecules, screening of immunoglobulinlibraries (Orlandi D. R. et al., 1989. Proc. Natl. Acad. Sci. U.S.A.86:3833-3837; Winter G. et al., 1991. Nature 349:293-299) or generationof monoclonal antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the Epstein-Barr virus(EBV)-hybridoma technique (Kohler G. et al., 1975. Nature 256:495-497;Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote R J. et al.,1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030; Cole S P. et al.,1984. Mol. Cell. Biol. 62:109-120).

In cases where target antigens are too small to elicit an adequateimmunogenic response when generating antibodies in-vivo, such antigens(haptens) can be coupled to antigenically neutral carriers such askeyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serumalbumine (BSA)] carriers (see, for example, U.S. Pat. Nos. 5,189,178 and5,239,078]. Coupling a hapten to a carrier can be effected using methodswell known in the art. For example, direct coupling to amino groups canbe effected and optionally followed by reduction of the imino linkageformed. Alternatively, the carrier can be coupled using condensingagents such as dicyclohexyl carbodiimide or other carbodiimidedehydrating agents. Linker compounds can also be used to effect thecoupling; both homobifunctional and heterobifunctional linkers areavailable from Pierce Chemical Company, Rockford, Ill. The resultingimmunogenic complex can then be injected into suitable mammaliansubjects such as mice, rabbits, and the like. Suitable protocols involverepeated injection of the immunogen in the presence of adjuvantsaccording to a schedule which boosts production of antibodies in theserum. The titers of the immune serum can readily be measured usingimmunoassay procedures which are well known in the art.

The antisera obtained can be used directly or monoclonal antibodies maybe obtained as described hereinabove.

Antibody fragments can be obtained using methods well known in the art.[(see, for example, Harlow and Lane, “Antibodies: A Laboratory Manual”,Cold Spring Harbor Laboratory, New York, (1988)]. For example, antibodyfragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g., Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. As describedhereinabove, an (Fab′)2 antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages to produce 3.5S Fab′ monovalentfragments. Alternatively, enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. Ample guidancefor practicing such methods is provided in the literature of the art(for example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647; Porter, R R., 1959. Biochem. J. 73:119-126). Other methods ofcleaving antibodies, such as separation of heavy chains to formmonovalent light-heavy chain fragments, further cleavage of fragments,or other enzymatic, chemical, or genetic techniques may also be used, solong as the fragments bind to the antigen that is recognized by theintact antibody.

As described hereinabove, an Fv is composed of paired heavy chainvariable and light chain variable domains. This association may benoncovalent (see, for example, Inbar et al., 1972. Proc. Natl. Acad.Sci. USA. 69:2659-62). Alternatively, as described hereinabove thevariable domains can be linked to generate a single chain Fv by anintermolecular disulfide bond, or alternately, such chains may becross-linked by chemicals such as glutaraldehyde.

Preferably, the Fv is a single chain Fv.

Single chain Fv's are prepared by constructing a structural genecomprising DNA sequences encoding the heavy chain variable and lightchain variable domains connected by an oligonucleotide encoding apeptide linker. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two variable domains. Ample guidancefor producing single chain Fv's is provided in the literature of the art(for example, refer to: Whitlow and Filpula, 1991. Methods 2:97-105;Bird et al., 1988. Science 242:423-426; Pack et al., 1993.Bio/Technology 11:1271-77; and Ladner et al., U.S. Pat. No. 4,946,778).

Isolated complementarity determining region peptides can be obtained byconstructing genes encoding the complementarity determining region of anantibody of interest. Such genes may be prepared, for example, by RT-PCRof mRNA of an antibody-producing cell. Ample guidance for practicingsuch methods is provided in the literature of the art (for example,refer to Larrick and Fry, 1991. Methods 2:106-10).

It will be appreciated that for human therapy, humanized antibodies arepreferably used. Humanized forms of non human (e.g., murine) antibodiesare genetically engineered chimeric antibodies or antibody fragmentshaving—preferably minimal—portions derived from non human antibodies.Humanized antibodies include antibodies in which complementarydetermining regions of a human antibody (recipient antibody) arereplaced by residues from a complementarity determining region of a nonhuman species (donor antibody) such as mouse, rat or rabbit having thedesired functionality. In some instances, Fv framework residues of thehuman antibody are replaced by corresponding non human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported complementaritydetermining region or framework sequences. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thecomplementarity determining regions correspond to those of a non humanantibody and all, or substantially all, of the framework regionscorrespond to those of a relevant human consensus sequence. Humanizedantibodies optimally also include at least a portion of an antibodyconstant region, such as an Fc region, typically derived from a humanantibody (see, for example, Jones et al., 1986. Nature 321:522-525;Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op.Struct. Biol. 2:593-596).

Methods for humanizing non human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non human. These non humanamino acid residues are often referred to as imported residues which aretypically taken from an imported variable domain. Humanization can beessentially performed as described (see, for example: Jones et al.,1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988. Science 239:1534-1536; U.S. Pat. No. 4,816,567)by substituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanized antibodies are chimeric antibodies, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non human species. In practice, humanizedantibodies may be typically human antibodies in which somecomplementarity determining region residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [see, for example, Hoogenboomand Winter, 1991. J. Mol. Biol. 227:381; Marks et al., 1991. J. Mol.Biol. 222:581; Cole et al., “Monoclonal Antibodies and Cancer Therapy”,Alan R. Liss, pp. 77 (1985); Boerner et al., 1991. J. Immunol.147:86-95). Humanized antibodies can also be made by introducingsequences encoding human immunoglobulin loci into transgenic animals,e.g., into mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon antigenic challenge, humanantibody production is observed in such animals which closely resemblesthat seen in humans in all respects, including gene rearrangement, chainassembly, and antibody repertoire. Ample guidance for practicing such anapproach is provided in the literature of the art (for example, referto: U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126,5,633,425, and 5,661,016; Marks et al., 1992. Bio/Technology 10:779-783;Lonberg et al., 1994. Nature 368:856-859; Morrison, 1994. Nature368:812-13; Fishwild et al., 1996. Nature Biotechnology 14:845-51;Neuberger, 1996. Nature Biotechnology 14:826; Lonberg and Huszar, 1995.Intern. Rev. Immunol. 13:65-93).

In order to identify putative agents capable of specifically regulatingMMP-9, an agent may be assessed regarding its capability of interactingwith the OG domain of MMP-9.

Thus, according to an aspect of the invention, there is provided amethod to determine whether an agent is a specific regulator of MMP-9comprising determining whether the agent is capable of interacting withan OG domain of MMP-9, the agent being a putative MMP-9 specificregulator.

Through meticulous experimentation, the present inventors have uncoveredthe 3D structure of full-length pro-MMP-9. The complete MMP-9 3Dstructure described herein may be used in the rational design of drugswhich modulate (preferably inhibit) the action of MMP9. These MMP9modulators may be used to prevent or treat the undesirable physical andpharmacological properties of MMP9 activity. Thus, according to oneembodiment of this aspect of the invention, an agent may be assessed forits ability to specifically regulate MMP-9 by comparing its structurewith the structure of the MMP-9 OG domain. This may be performed byusing computer models of the full length MMP-9 such as those generatedby the present inventors with the aid of programs such as GASBOR andCHADD. This method may be particularly suitable for identifying peptideagents and small molecules.

Once the structure of the agent is at hand one can design peptides whichfit into the 3D structure of the OG domain and hopefully stanilize ordisrupt it. Such peptides/small molecule can be screened forspecifically binding the OG domain.

Generation of peptide mimetics, as described hereinabove, may beeffected using various approaches, including, for example, displaytechniques.

Methods of constructing such display libraries are well known in theart. Such methods are described in, for example, Young A C, et al., “Thethree-dimensional structures of a polysaccharide binding antibody toCryptococcus neoformans and its complex with a peptide from a phagedisplay library: implications for the identification of peptidemimotopes” J Mol Biol 1997 Dec. 12; 274(4):622-34; Giebel L B et al.“Screening of cyclic peptide phage libraries identifies ligands thatbind streptavidin with high affinities” Biochemistry 1995 Nov. 28;34(47):15430-5; Davies E L et al., “Selection of specific phage-displayantibodies using libraries derived from chicken immunoglobulin genes” JImmunol Methods 1995 Oct. 12; 186(1):125-35; Jones C R T al. “Currenttrends in molecular recognition and bioseparation” J Chromatogr A 1995Jul. 14; 707(1):3-22; Deng S J et al. “Basis for selection of improvedcarbohydrate-binding single-chain antibodies from synthetic genelibraries” Proc Natl Acad Sci USA 1995 May 23; 92(11):4992-6; and Deng SJ et al. “Selection of antibody single-chain variable fragments withimproved carbohydrate binding by phage display” J Biol Chem 1994 Apr. 1;269(13):9533-8, which are incorporated herein by reference.

Peptide mimetics can also be uncovered using computational biology. Forexample, various compounds can be computationally analyzed for anability to bind OG domain using a variety of three-dimensionalcomputational tools as described in the Examples section herein below.Software programs useful for displaying three-dimensional structuralmodels, such as RIBBONS (Carson, M., 1997. Methods in Enzymology 277,25), O (Jones, T A. et al., 1991. Acta Crystallogr. A47, 110), DINO(DINO: Visualizing Structural Biology (2001) www.dino3d.org); andQUANTA, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP(reviewed in Kraulis, J., 1991. Appl Crystallogr. 24, 946) can beutilized to model interactions between the OG domain and prospectivepeptide mimetics to thereby identify peptides which display the highestprobability of binding to the OG region. Computational modeling ofprotein-peptide interactions has been successfully used in rational drugdesign, for further detail, see Lam et al., 1994. Science 263, 380;Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993.Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993.Perspectives in Drug Discovery and Design 1, 109, and Mauro M J. et al.,2002. J Clin Oncol. 20, 325-34.

According to another embodiment of this aspect of the invention, anagent may be assessed for its ability to specifically regulate MMP-9 byincubating the agent with isolated MMP-9. Since the amino acid sequenceof MMP-9 is known, the isolated MMP-9, or fragment thereof whichcomprises the OG domain may be generated using standard recombinant DNAtechnology or by chemical synthesis. Standard protein labelingtechniques may be used for assaying binding of the agent to the target.Labeling may be direct (e.g., by S35 labeling of the MMP-9) orindirectly, such as by the use of secondary antibodies. Standardimmunological (ELISA, immunoprecipitation) and biochemical (e.g., gelfiltration) methods can be used for assessing agent binding.

Once putative agents are identified, they may be assayed for theirabilities to regulate MMP-9 functions and for their ability to beselective towards MMP-9. An example of such an assay is the in-situzymographic analysis of collagenolytic activity described in Example 7herein below.

As mentioned, MMP-9 is known to be a prototypical target in inflammatorydiseases, because of its tissue-damaging roles andinflammation-promoting processing of soluble proteins, includingprotease inhibitors, chemokines and cytokines. Therefore, agents capableof down-regulating activities of MMP-9 may be used to treat MMP-9related disorders.

Thus, according to an aspect of the invention, there is provided amethod of treating a MMP-9 mediated medical condition, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of an agent which specifically interacts with an OGdomain of MMP-9, thereby treating the MMP-9 mediated medical condition.

As used herein the term “subject in need thereof” refers to a mammal,preferably a human subject.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of an MMP-9 mediated disease or condition.

The phrase “MMP-9 mediated medical condition” refers to a disease ordisorder in which MMP-9 may be attributed to its onset or progression.An example of an MMP-9 mediated medical condition is cancer, e.g.metastatic cancer such as breast, ovarian, bone, lung, pancreatic andprostate cancer.

In addition to playing a role in cancer, MMP-9 may be involved in otherpathologies, for example, in arthritis or in neurodegenerative diseasessuch as multiple sclerosis (Firestein, Curr. Opin. Rheumatol. 4:348-354(1992); Gijbels et al., J. Neuroimmunol. 41:29-34 (1992)). For example,high levels of MMP-9 have been detected in serum and synovial fluid ofpatients with inflammatory arthritis such as rheumatoid arthritiscompared to healthy patients or patients with osteoarthritis (Ahrens etal., Arthritis & Rheumatism 39:1576-87 (1996); Gruber et al., Clin.Immunol. & Immunopathol., 78:161-171 (1996)). In addition, a correlationhas been reported between the arthritic activity score of a joint andthe amount of MMP-9 in the aspirated synovial fluid (Koolwijk et al. J.Reumatology, 22:385-393 (1995)).

Expression of MMP-9 is also detected in diseases of the nervous system.For example, prominent expression of MMP-9 has been found in reactiveastrocytes and macrophages in demyelinating lesions compared to normalbrain tissue (Cuzner et al., J. Neuropathol. Exp. Neurol, 55:1194-1204(1996)). MMP-9 is elevated in encephelomyelitis (Gijbels, et al., J.Neuro. Res. 36:432-440 (1993); Proost, et al., Biochem, Biophys, Res.Comm. 192:1175-1181 (1993)), in the cerebrospinal fluid of patients withmultiple sclerosis (Leppert, et al., Brain 121:2327-2334 (1998);Rosenberg et al., Brain Res., 703:151-155 (1995)), and in patients withAIDS-related dementia (Conant, et al., Annals of Neurology 46: 391-398(1999)). Furthermore, in patients with amyotrophic lateral sclerosis,MMP-9 expression is found in the pyramidal neurons of the motor cortexand in the motor neurons of the spinal cord (Lim et al., J. Neurochem.,67:251-259 (1996)).

MMP-9 has also been associated with a variety of other inflammatorydiseases. For example, a high level of MMP-9 activity is found in thevessel wall of aortic aneurysms (Freestone, et al. Arteriosclerosis,Thrombosis & Vascular Biology, 15:1145-1151 (1995); Newman et al.,Connective Tissue Research, 30:265-276, (1994); Sakalihasan et al., J.Vascular Surgery, 24:127-33 (1996)). In addition, patients with giantcell arteritis have increased levels of MMP-9, and MMP-9 mRNA is foundin smooth muscle cells and fibroblasts in the regions of fragmentedelastic tissue in the lamina media of inflammed vessels (Sorbi, et al.,Arthitis & Rheumatism, 35:1747-1753 (1996)). Increased levels of MMP-9are also found in sputum of patients with cystic fibrosis and inbronchoalveolar lavage fluids of those with bronchiectasis (Delacourt etal., Amer. J. Respiratory & Critical Care Med., 152:765-764 (1995);Sepper et al, Chest, 106:1129-1133 (1994)). High levels of MMP-9 havealso been found in blister fluids from the skin lesions of bullouspemphigoid patients (Stahle-Backdahl et al., J. Clinical Invest.,93:2022-2030 (1994)).

MMP-9 expression has also been implicated in the pathogenesis of severalother diseases. For example, MMP-9 has been implicated in polycystickidney disease (Murray et al., Conn. Tissue Res., 33:249-256 (1996)),membranous nephropathy (McMillin et al., J. Clin. Invest., 97:1094-1101(1996)), and Alzheimer's disease (Lim et al., J. Neurochem.,68:1606-1611 (1997)).

Accordingly, the present invention contemplates the treatment of all theabove referred to diseases or conditions using agents capable ofselectively interacting with the OG domain of MMP-9.

The agents of the present invention can be administered to the subjectper se, or as part of a pharmaceutical composition, which also includesa physiologically acceptable carrier. The purpose of a pharmaceuticalcomposition is to facilitate administration of the active ingredient toan organism.

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

As used herein, the term “active ingredient” refers to the agentaccountable for the intended biological effect i.e. down-regulation ofan activity of MMP-9.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. One of the ingredients included in thepharmaceutically acceptable carrier can be for example polyethyleneglycol (PEG), a biocompatible polymer with a wide range of solubility inboth organic and aqueous media [Mutter et al. (1979)].

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer a preparation in a local rather thansystemic manner, for example, via injection of the preparation directlyinto a specific region of a patient's body.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., a sterile, pyrogen-free,water-based solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, for example, conventional suppository bases such as cocoabutter or other glycerides.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., a nucleic acid construct) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Dosage amount and administration intervals may be adjusted individuallyto provide sufficient plasma or brain levels of the active ingredient toinduce or suppress the biological effect (i.e., minimally effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

GENERAL MATERIALS AND METHODS

Expression of MMP-9: Recombinant pro-MMP-9 was expressed by infection ofSf9 insect cells with a baculovirus carrying the cDNA of human proMMP-9[19]. Liter quantities of cell culture fluids were centrifuged, filteredand purified to homogeneity by gelatin-Sepharose chromatography [52].The material was extensively dialyzed in 100 mM Tris pH 7.4, 100 mMNaCl, 10 mM CaCl₂ (buffer C) before further processing and about 20 mgwere used in the present study. A mutant lacking the OG-domain(MMP-9ΔOG) was prepared in a similar way [19].

Small Angle X-ray Scattering: SAXS experiments in solution wereperformed at station 2.1 [53] of the Synchrotron Radiation Source,Daresbury Laboratory, UK, following standard procedures. The proteinsolution was centrifuged for 5 min at 13,000×g before being measured at4° C. Scattering curves were collected with a two-dimensional multiwireproportional counter, at sample-to-detector distances of 1 m (7 mg/ml,100 μl) and 4.25m (0.8, 1.6, 2.5 mg/ml, 100 μl), at a wavelength (λ) of1.54 Å, covering the momentum transfer range 0.008<q<0.78 Å⁻¹ (q=4π sinθ/λ, where 2θ is the scattering angle). The data were collected in 30successive 1 minute frames, and then normalized to the intensity of theincident beam, radially integrated over a 60° sector, averaged over theframe number and normalized to the detector response. The scattering ofthe buffer was then subtracted and the low- and high-angle curves weremerged over the q range of 0.05-0.15 Å⁻¹. Reproducibility of theintensity as a function of time was evidence for lack of radiationdamage of the monomeric pro-MMP-9 sample. The radius of gyration (R_(g))was evaluated using the Guinier approximation: I(q)=I(0)exp(−q²R_(g)²/3) for qR_(g)<1.3 [54], and also from the entire scattering curve withthe indirect Fourier-transform program GNOM [55]. GNOM also provides thedistance distribution function p(r) of the particle and its maximumdimension D_(max), defined as the point where p(r) becomes zero. Todetermine p(r), p(0)=0 and p(D_(max)) were assigned free, in the firstinstance, to judge whether the chosen r interval was correct. D_(max)was the lowest value yielding the lowest positive p(D_(max)). Afterfixing D_(max), p(0) and p(D_(max)) were fixed to zero. The data wasthen cut at the low and high angle region until the p(r) functionsconverged.

The crystal structures of the two MMP-9 domains (N-terminal catalyticdomain and C-terminal hemopexin-like domain) were analyzed using theprogram CRYSOL [36] in order to calculate their correspondingtheoretical scattering curves. These were further Fourier-transformed toyield their theoretical pair distribution functions, while the D_(max)and R_(g) values were calculated. Ab initio modeling of the SAXS curvesare described in detail below. Structural figures were made with PyMOL(DeLano, W. L. The PyMOL Molecular Graphics System (2002) DeLanoScientific, San Carlos, Calif., USA. www.pymol.org).

To further confirm the accuracy of the model, its solution hydrodynamicproperties were calculated using HYDROPRO [34], and then compared to theexperimental values. The radius of shell minibeads was varied from 2.2to 4.2 Å in six increments. The solvent density and viscosity, and theprotein partial specific volume were calculated using SEDNTERP [27].Sphere radii for the hydrodynamic shell model varied between 3.8 to 5.3Å. The radius of the dummy residues (DRs) in the SAXS model is 3.8 Å.However, the actual dimension of the shell model is slightly bigger dueto protein hydration, yet the extent of enlargement is difficult todetermine [34]. It was previously suggested that increasing the DRsradius by 1.5 Å would take into account hydration reliably [56].

Atomic Force Microscope imaging: Imaging was performed using a Multimodeatomic force microscope (MMAFM Veeco/Digital Instruments, Santa Barbara,Calif., USA) equipped with an E-scanner, with a maximum scan range of14×14 μm². Samples were imaged in air or in buffer using Tapping Mode.To obtain samples free of artifacts from spurious adsorbates, mainlysalt deposition, an aggressive rinsing procedure was required. By usingthe amine-modified silane surfaces and cross-linking procedure, it waspossible to prepare surfaces that removed nearly all the background (asdetermined by blank runs), while maintaining significant surfaceconcentration of protein.

In order to minimize the amount of force applied, the amplitude setpoint was adjusted to the maximum value that gave a stable trace.High-resolution images of biological samples in air were obtained using“spike” tips-DP14 “HI′RES™” probes from Mikromasch (Estonia). Theseprobes have a resonant frequency of ca. 160 kHz, a force constant of ca.5 N/m, and a rated radius of curvature of 1 nm or less, but are onlysuitable for measurements on surfaces with rms roughness less than 20 nmdue to the presence of additional “spikes” which could cause multiplecontacts on a rough surface DNP-S probes (Veeco) with nominal radius of20 nm were used for the liquid measurements, which were performed in thestandard MMAFM liquid cell. The sizes of the protein molecules weredetermined from cross-sectional analysis. The width values were thancorrected for broadening by the tip, by subtracting the tip envelope asobserved from a typical high resolution SEM image.

Size exclusion chromatography: The oligomeric mixture of pro-MMP-9 wasloaded on a Superdex-200 column (300×10 mm, Amersham Biosciences),pre-equilibrated and operated at 4° C. The sample volume was 100 μl of1.1 mg/ml pro-MMP-9 and the flow rate was 0.5 ml/min. Elution wasmonitored by absorbance at 280 nm. The Stokes radius was determined byanalysis of the elution time with respect to a calibration curve usingboth a Porath plot and a Laurent and Killander plot [Siegel, L. M., andMonty, K. J. (1966). Biochim Biophys Acta 112, 346-362]. The fivestandard proteins (Amersham Biosciences) of known Stokes radii used forthe calibration curve were thyroglobulin (85 Å), ferritin (61 Å nm),catalase (52.2 Å), aldolase (48.1 Å), and albumin (35.5 Å). The voidvolume measured by blue dextran had a retention time, t₀ of 16.23 minand the total volume determined by vitamin B-12 had a retention time,t_(T), of 39.44 min. From these values, the partition coefficient of agiven protein, Kd, was calculated as: Kd=(t_(e)−t₀)/(t_(T)−t₀), t_(e)being the retention time of a given protein. Very similar results wereobtained for both the Porath and Laurent and Killander plots. Theuncertainties in retention times for three repeating sets of experimentswere 0.5% on average. The linear least-square fits had a correlationcoefficient of r²=0.97 for both the Porath and Laurent and Killanderplots.

Glycerol-gradient sedimentation: A sample (0.2 mg) of purified pro-MMP-9was layered onto four polyallomer tubes containing 10-45% glycerolgradient (prepared in GradientMaster BioComp™) in buffer. The tubes werethen centrifuged in a SW41 rotor at 37,000 rpm, 63 h, 4° C. The gradientwas then fractionated to 0.5 ml samples that were assayed for thepresence of monomeric and other oligomeric structures by gelatinzymography [Masure, S., Proost, P., Van Damme, J., and Opdenakker, G.(1991). Eur J. Biochem. 198, 391-398]. Fractions containing homogenousmonomeric structures were pooled and dialyzed against buffer to removeexcess glycerol. Protein concentration was determined using the BCAprotein assay kit (Pierce).

Analytical ultracentrifugation: Sedimentation velocity experiments wereperformed in a Beckman Optima XL-A analytical ultracentrifugationequipped with An-50 Ti rotor. Experiments were carried at 20° C. inbuffer C. A sample at a protein concentration of 0.4 mg/ml was loadedinto 12 mm path cells and centrifuged at 50,000 rpm. Absorbance at 280nm was recorded every 160 sec using 0.001 cm radial spacing over theradial range 6-7.3 cm.

The sedimentation profiles were analyzed using the software SEDFIT[Schuck, P. (2000). Biophys J. 78, 1606-1619] which allows evaluation ofthe sedimentation coefficient (s). The 130 experimental curves wereanalyzed and the distribution of the sedimentation coefficient, c(s),was obtained between 0.3 to 50 S at a resolution of 200 steps at thisregion, and grid size of 500 points. The confidence level for theparameterization was set to 0.9. The software SEDNTERP [Laue, T. M.,Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992). AnalyticalUltracentrifugation in Biochemistry and Polymer Science (Cambridge,U.K.: Royal Society of Chemistry)] was used to estimate the solventdensity (ρ) to be 1.0062 gr/cm³, and the viscosity (η) to be 1.045 cP.The protein partial specific volume (υ) was calculated based on theamino acid and glycan composition to be 0.7328 cm³/gr, where the aminoacid parameters by Kharakoz [Kharakoz, D. P. (1997). Biochemistry. 36,10276-10285] were used.

Amine Functionalized Substrate Preparation for AFM imaging: Thesesurfaces were selected for their high concentration of primary aminegroups that interact with glutaraldehyde which cross-links and binds theprotein. Glutaraldehyde forms an amide linkage to an amine surfacegroup. The free amine of glutaraldehyde crosslinks the proteinspontaneously by covalent interaction to amine groups ubiquitouslypresent on the outer surface of the protein. Thus, there is no need forengineering or modification in order to affix the protein to thesurface. Furthermore, this method only minimally perturbs the randomdistribution in protein conformation and orientation on the surface.

Preparation and characterization of the amine functionalized substratesused to specifically bind the protein (Veeco Metrology, Inc SantaBarbara, Calif. Part Number FSUB-11) is described briefly. Silicon chipsof 1 cm² were diced from polished <111> wafers (International WaferServive INC.-Denmark). The silicon chip was modified with an amineterminated silane by plasma enhanced chemical vapor deposition (4^(th)State, LLC, Belmont, Calif.) to create an amine functionalizedsubstrate. The surface composition of the substrates was analyzed byX-ray Photoelectron Spectroscopy (XPS) using a Kratos Axis Ultra(Kratos, Manchester, UK) with a monochromated Al k_(α) x-ray source at1486.6 eV both before and after amine functionalization. Amine groups,indicated by a nitrogen peak in the XPS spectra, were present at thesurface only after the amine functionalization. Root mean square (RMS)surface roughness of the treated substrates was 1.8 Å as determined byAtomic Force Microscopy (AFM) applying tapping mode, in air withOTESPprobes (VEECO). A Horseradish Peroxidase (HRP) assay was used todetermine the binding potential of the amine functionalized substrates.HRP labeled antibodies were immobilized to the amine functionalizedsubstrate through a glutaraldehyde crosslinker. Substrates weresonicated to remove all unbound antibody and then analyzed with SureBlueReserve TMB 1 Component Peroxidase Substrate (Kirkegaard and Perry Labs,Maryland). Binding activity was determined by reading absorbance at 450nm.

Protein Immobilization Procedure: The aminized dies were kept in adesiccator at 4° C. Immediately before use, pro-MMP-9 was immobilized tothe amine functionalized substrates through a glutaraldehyde crosslinkerby the following procedure: 1.25% glutaraldehyde in 0.1 M SodiumCarbonate solution (pH 9) was incubated on the amine functionalizedsubstrate overnight. The substrate was then rinsed thoroughly withsodium carbonate solution to remove unbound glutaraldehyde. Samplevolume of 100 μl containing 0.1 mg/ml monodisperse solution ofpro-MMP9ΔOG mutant or wild-type pro-MMP-9, fractionated to contain themonomeric form, were then incubated on the dies for 3 h. The sampleswere rinsed gently with 2×200 μl buffer followed by 5×200 μl Milli-Qwater and finally dried under a stream of nitrogen. The glutaraldehydeserved as covalent cross-linker of the protein to the aminized surface.This ensured robust attachment of the protein molecules to the surfaceduring the rinsing and subsequent imaging. For AFM experiments run inbuffer, the samples were kept hydrated continuously.

Ab Initio modeling of SAXS curves: The programs GASBOR [Svergun, D. I.,Petoukhov, M. V., and Koch, M. H. (2001). Biophys J 80, 2946-2953] andCHADD [Petoukhov, M. V., Eady, N. A., Brown, K. A., and Svergun, D. I.(2002). Biophys J 83, 3113-3125] were used to generate low-resolutionmodels. To take into account the glycosylations on pro-MMP-9 it wasassumed that a single glycan is equivalent to ˜1.6 amino acid residuesaccording to its electronic density and length [Receveur, V., Czjzek,M., Schulein, M., Panine, P., and Henrissat, B. (2002). J Biol. Chem.277, 40887-40892]. It was also found that this value represents therelation between the averaged molecular weights of glycans and residues.The total number of DRs was than calculated, based on the amino acidsequence and the glycan composition that was previously characterized[Van den Steen, P. E., Van Aelst, I., Hvidberg, V., Piccard, H., Fiten,P., Jacobsen, C., Moestrup, S. K., Fry, S., Royle, L., Wormald, M. R.,Wallis, R., Rudd, P. M., Dwek, R. A., and Opdenakker, G. (2006). J Biol.Chem. 281, 18626-18637].

Several models of the same input parameters were generated for eachmethod, in order to check for convergence of the model to a uniquesolution through the random Monte-Carlo fitting procedure. The modelswere inspected using DAMAVER [Volkov, V. V., and Svergun, D. I. (2003).Journal of Applied Crystallography 36, 860-864] in order to choose themost probable solution, and to compute averaged normalized spatialdiscrepancy (NSD) values (see Supplemental Data Results). The crystalstructures of the terminal domains [Elkins, P. A., Ho, Y. S., Smith, W.W., Janson, C. A., D'Alessio, K. J., McQueney, M. S., Cummings, M. D.,and Romanic, A. M. (2002). Acta Crystallogr D Biol Crystallogr 58,1182-1192; Cha, H., Kopetzki, E., Huber, R., Lanzendorfer, M., andBrandstetter, H. (2002). J Mol Biol 320, 1065-1079] were then docked inthe representative model using the software SUPCOMB [Kozin, M. B., andSvergun, D. I. (2001). Journal of Applied Crystallography 34, 33-41].

In-situ zymographic analysis of collagenolytic activity: In-situzymography [Deshane, 2003] was performed by incubation of humanfibrosarcoma HT1080 (CCL-121; ATCC, Rockville, Md.) cells with 60 nManti-MMP9 hr or corresponding buffer for control, and 450 nM labeledcollagen (Oregon green-labeled collagen type-IV that is intramolecularlyquenched-Molecular probes) at 37° C. for 16 h. The degradation ofcollagen gives rise to green fluorescence, which is indicative of netcollagenolytic activity. Prior to imaging, the samples were stained withHoechst 33258 (Molecular Probes) at a final concentration of 3.8 μg/mlfor nuclear labeling. Samples were examined and photographed byfluorescence microscope (E600; Nikon, Tokyo, Japan) equipped with PlanFluor objectives connected to a CCD camera (DMX1200F; Nikon). Theexperiment was repeated six times. Images were assembled using AdobePhotoshop (Adobe systems, San Jose, Calif.).

Example 1 Isolation and Characterization of pro-MMP-9 in its MonomericForm

Molecular size and shape determination, structural reconstruction, andanalysis of single molecule images of pro-MMP-9 monomers requiremonodispersed and homogeneous protein samples. The following exampledescribes a combination of various methods to express, isolate andcharacterize the monomeric form of pro-MMP-9. Characterization ofmolecular radius was used to validate spectroscopic shape determination.

Results

Recombinant pro-MMP-9 was expressed and purified from baculovirusinfected Sf9 cells as previously reported [19] (see Materials andMethods). This enzyme forms mixtures of monomers and other higheroligomeric species [20].

FIG. 1B shows the relative molecular ratio of pro-MMP-9 monomer to itsoligomeric species as determined by analytical size exclusionchromatography (SEC). The main peak (No. 3) in the chromatogramcomprises the pro-MMP-9 monomer with Stokes radius of 45.4 Å (seeinset). The Stokes radius was determined based on the correspondingretention time using conventional procedures.

Isolation of pro-MMP-9 monomers from higher oligomeric species inpreparative amounts was achieved by glycerol-gradient sedimentation[26]. FIG. 1C shows zymography analysis of the various fractions. Theisolated monomer fraction was subjected to analyticalultracentrifugation (AUC) for additional estimation of its Stokes radius(FIG. 1D). In this sedimentation velocity experiment, a uniformpro-MMP-9 solution is subjected to a gravimetric field. This produces adepletion of solute near the meniscus and the formation of a sharpboundary between the depleted region and the uniform concentration ofsedimenting solute (FIG. 1D, inset). The rate of movement of thisboundary can be measured and leads to the determination of thesedimentation coefficients, which depends directly on the mass of theparticles and inversely on the frictional ratio, which is in turn ameasure of effective size and shape.

Pro-MMP-9 monomer was found to sediment as a single species with themain peak representing 91% of the total protein in the sample with anormalized sedimentation coefficient, s°_(20,w), of 4.4 S (FIG. 1D).This value is compatible with a recent measurement [19]. A Stokes radiusof 44.1 Å was computed by the program SEDNTERP [27] using a calculatedpartial specific volume of 0.7328 cm³/g. AUC-based shape analysis (usingthe experimental frictional ratio) was indicative of elliptical shapewith axial ratio a/b of 1:6. The molecular radial results obtained byAUC are consistent with the value obtained by SEC (45.4 Å). In addition,theoretical estimation of the radius, by SEDNTERP, using the pro-MMP-9molecular mass and amino acid and glycan composition yielded a molecularequivalent spherical radius of 28.7 Å. Deviation of this value from theexperimental Stokes radii provides another indication for anon-spherical shape, either elongated or incorporating cavities.

Example 2 Molecular Shape Analysis of pro-MMP-9 by Small-Angle X-rayScattering (SAXS) Reveals an Elongated Three-Domain Structure

Results

The global conformation of pro-MMP-9 monomer in solution wasinvestigated by SAXS. In SAXS, the scattering profile derives from theentire ensemble of randomly oriented molecules, yielding informationabout their averaged conformation (on the order of approximately ananometer). SAXS thus differs from crystallographic structural analysis,which requires hard-to-get high quality crystals of macromolecules, andis one of a few structural techniques for studying proteins in solution.This method utilizes the elastic scattering of incident X-ray photons bythe target molecule electrons. The electron density distribution, asgoverned by the arrangement of atoms in the molecule leads to aninterference pattern. The three dimensional shape of the molecule isthen reconstructed from the scattering profile [28].

Scattered intensity was observed over a momentum transfer range0.008<q<0.46 Å⁻¹ corresponding to a d-spacing range of 14<r<785 Å (FIG.2A). The lower value (14 Å) fixes the ultimate resolution obtainable inthe measurements. The scattered intensities are linear in the small-qregion (scattering profile see FIG. 2A) and are nicely fitted by theGuinier law. The slope was found to be weakly correlated with proteinconcentration. This means that neither aggregation nor inter-particleinterference contribute significantly to the signal. The radius ofgyration (R_(g)) resulting from the measurement is 50±2.7 Å. Thefunction p(r) represents the distribution of interatomic distanceswithin the molecule (pair distribution function—FIG. 2A, inset).Extraction of R_(g) from p(r) gives a comparable value, 49.2 Å,indicating accurate preliminary data analysis (prior to the fittingprocedure). The maximal interatomic distance (D_(max)) is 160 Å. Theshape of p(r) is indicative of an elongated ellipsoid structure (see forexample [29-31]).

The three dimensional reconstruction model of pro-MMP-9 was obtainedusing the programs GASBOR [32] and CHADD [33]. Theoretical scatteringcurves are simulated from three-dimensional arrangements of sphericalcenters (or dummy residues) representing protein residues, which combineto form the overall protein shape. The final protein shape is determinedby iterative fitting of the simulated theoretical curves to theexperimental data. The advantage of CHADD is in the use of a prioriknowledge obtained from available crystal structure of isolated domainsto introduce constrains in the data analysis procedures. In contrast,models produced by GASBOR are calculated without any a priori knowledge.A detailed comparison between CHADD and GASBOR is described in Example3, herein below.

FIG. 2B shows the three-dimensional reconstructed structure ofpro-MMP-9. The Stokes radius of the reconstructed structure of pro-MMP-9was calculated using the program HYDROPRO [34]. This calculated radiusranges between 44.9 and 47.1 Å, which agrees with the measured values of45.4 and 44.1 Å obtained by SEC and AUC, respectively. Furthermore, boththe SAXS model and axial ratio parameter obtained by AUC, suggest anelongated shape. Hence, the reconstructed shape restored from theexperimental SAXS profile is consistent with the measured hydrodynamicdata obtained both by SEC and AUC.

The simulated curve fitting analysis of this structure is presented inFIG. 2A (gray curve). The location of the alpha carbon backbone of thepro-catalytic domain was used as a constraint in the structuralreconstruction analysis while the OG and the hemopexin-like domains werereconstructed using CHADD. Finally, the crystal structures of thepro-catalytic [22] domain and the hemopexin-like domain [24] weresequentially docked to the contour density using the software SUPCOMB[35] (FIG. 2B). The remaining density belongs to the OG domain thatseparates the two terminal domains by ˜30 Å. This value was furtherverified by calculating the theoretical p(r) curves based on the crystalstructures of the isolated N-terminal and C-terminal domains using thesoftware CRYSOL [36]. The calculated D_(max) values for these domainsare 80 Å and 50 Å, respectively. Subtracting these values from theexperimental D_(max) of full-length pro-MMP-9 (160 Å), provides furtherverification of the reconstructed structure where the terminal domainsare separated by ˜30 Å.

Inspecting the volumes occupied by the OG and hemopexin-like domainsreveals that they are of similar volume. However, the calculatedmolecular weight of the OG domain, including the O-glycans, is abouthalf of the hemopexin-like domain. Computational sequence analysis ofthe OG domain using PONDR [37] revealed that this region issignificantly disordered relative to the other domains (FIG. 2C). Thus,despite its observed compact conformation, this proline-rich OG domainpossesses a disordered structure of relatively low density. Therefore,the relatively bulky electron density of the OG domain, detected bySAXS, represents a range of conformations retained by this linkerpeptide in solution. This suggests that the OG linker domain isflexible. The structural modeling program RAPPER [38, 39] was used tomodel possible linker conformations that will fit the observedscattering profile and density map. Specifically, 8 out of 500calculated conformers fit the SAXS model of the linker. Theoreticalscattering curves were calculated (using the SAXS program CRYSOL [36])for the overall pro-MMP-9 model structure. FIGS. 2D and 2E describes thebest linker models that fit both the experimental scattering curve andthe SAXS density map of pro-MMP-9. The OG linker appears to exhibitmultiple putative unstructured conformations.

Example 3 Comparison of Modeling Software for the Analysis of the SAXSData

Results

The programs GASBOR [Svergun, D. I., Petoukhov, M. V., and Koch, M. H.(2001). Biophys J 80, 2946-2953] and CHADD [Petoukhov, M. V., Eady, N.A., Brown, K. A., and Svergun, D. I. (2002). Biophys J 83, 3113-3125]were used to generate a low-resolution model of pro-MMP-9. Both programsfind a representation of the protein by a 3D arrangement of sphericalscattering centers that reproduce the measured scattering curve. Theadvantage of CHADD is in the use of a priori knowledge of a portion ofthe Cα locations as determined by the crystal structure of isolateddomains while the rest of the molecule is modeled, whereas GASBOR modelsthe whole structure without the incorporation of a priori knowledge Ineach of the programs, several independent computations are compared toanalyze the convergence of the solution structures.

Eight independent GASBOR runs converged to a unique solution of anelongated overall conformation where one end is of larger size and canharbor the crystal structure of the N-terminal domain whereas the otherend assumes a disk-like shape that can accommodate the crystal structureof the C-terminal domain (FIG. 5A). The terminal domains were docked tothe GASBOR model using the program SUPCOMB [Kozin, M. B., and Svergun,D. I. (2001). Journal of Applied Crystallography 34, 33-41]. Theremaining density belongs to the OG domain that separates the terminaldomains by 50 Å, taken as the OG domain length. The normalized spatialdiscrepancy (NSD) within the eight calculations ranged from 1.42 to1.57. The NSD value is a measure of the similarity between the solutionstructures: a lower value corresponds to a better overlap. The NSDvalues were calculated using DAMAVER [Petoukhov, M. V., and Svergun, D.I. (2003). Journal of Applied Crystallography 36, 540-544], which isalso capable of choosing the most probable solution (χ²=1.38) and todetermine the outliers.

In an alternate modeling scheme, which makes use of known structuralinformation, the full-length pro-MMP-9 structure was reconstructed usingCHADD. Eleven independent runs were computed and showed NSD values of1.59-1.75 with no outliers, suggesting convergence of the solutionstoward a unique model. The most probable solution (χ²=1.66) is shown inFIG. 5B. The model indicates an elongated, three-domain structure thatshows a large bimodal shape with disk-like domains connected by therelatively low-density OG domain. The crystal structures of thepro-catalytic domain and the hemopoxin domains were sequentially dockedto the contour density using the software SUPCOMB [Kozin, M. B., andSvergun, D. I. (2001). Journal of Applied Crystallography 34, 33-41].The remaining density belongs to the OG domain that separates the twoterminal domains by ˜30 Å, a value that well agrees with the theoreticalsize of the OG domain as computed by CRYSOL.

The two structures obtained by two independent modeling algorithms arethe result of convergence of several calculations to a unique solution.An NSD value of 1.68 was obtained for testing the similarity between theGASBOR and CHADD models, indicating good agreement between models.Although the GASBOR model is rather elongated (with OG domain of 50 Å asopposed to 30 Å) both models share very similar characteristics, i.e.,two fairly globular domains connected by a linker (elliptic N-terminaland C-terminal hemopexin joined by the OG linker) resulting in anoverall elongated structure composed from two fairly globular domainsconnected by a linker. The agreement of the size of the OG domain withthe value computed by CRYSOL, and the similarity in overall shape to theAFM results led the present inventors to choose CHADD as a more reliablemodel.

Example 4 Characterization of Shape and Domain Flexibility of pro-MMP-9by Single Molecule Imaging

Results

To further verify the SAXS analysis, the present inventors designed anexperiment to directly visualize the shape of pro-MMP-9 and evaluate themolecular properties of its OG domain as predicted in FIGS. 2B and E.Specifically, the present inventors conducted single molecule imaginganalysis of wild type and an OG-deleted mutant of pro-MMP-9(pro-MMP-9ΔOG) using atomic force microscopy (AFM). Reproducible imagesof single pro-MMP-9 molecules (FIG. 3) were obtained by cross linkingthe protein samples to an amine-modified silanized layer on a Si(111)surface prior to AFM imaging.

Samples were imaged both under buffer solution, and in air (FIGS. 6A-C).Best images were obtained in semi-dry mode using a “spike” tip. FIGS.3A-C show a single molecule image of wild type pro-MMP-9 immobilized onthe modified Si(111) surface. Consistent with the reported SAXSanalysis, the protein image possesses an elongated multi domainstructure. The image cross-section (FIG. 3C) representing height versuswidth reveals two separated protein domains presumably connected by theOG linker. In contrast, the pro-MMP-9ΔOG mutant lacking the 64-residueOG domain exhibits rather spherical shape with unresolved domainseparation (FIGS. 3D-F).

Example 5 Choice of Conditions and Controls for Atomic Force Microscopy(AFM) Imaging of pro-MMP-9

Results

Samples were imaged both under buffer solution, and in air. Although theformer mode approximates physiological conditions, the quality of theimages was poor (FIG. 6A). The poor quality of the images could arisefrom several factors: (1) The tips designed to be used for wetconditions had a significantly larger radius than the “spike” tips usedin ambient conditions. (2) The fully hydrated sample may be softer andeasier to distort under the tip pressure. (3) The binding of the proteinto the surface may still allow some degree of motion under fullyhydrated conditions. Therefore, in order to improve the image quality,ambient conditions were applied where the sample was rinsed and excesswater was removed by 2-3 minutes of gentle nitrogen flow. This procedurelikely leaves a thin hydration layer on the sample and is hence termed“semi-dry mode”. To check the protein shape in the absence of anyhydration layer, we applied thorough desiccation (FIG. 6B), which led toloss of fine features, contraction of the protein, and image noise inthe cross-sections.

The main difficulty in these semi-dry measurements was to obtain samplesfree of artifacts from spurious adsorbates, mainly salt deposition.Aggressive rinsing procedures were required to remove those adsorbates,but also led to the removal of much of the protein. By using theamine-modified silane surfaces and cross-linking procedure, it waspossible to prepare surfaces which removed nearly all the background (asdetermined by blank runs), while maintaining significant surfaceconcentration of protein, similar to what is seen under fully hydratedconditions. Blanks (FIG. 6C) prepared by incubation under identicalconditions as the samples, but with no pro-MMP-9 typically contain nomore than one feature on a 1 μm² image. Comparison to the samples withpro-MMP-9 indicated that on average less than 5% of the observedfeatures could have been artifacts due to debris or dried salt.

Statistical analysis of AFM images: Statistical analysis of height,width, and lobe-to-lobe distances were determined for wild typepro-MMP-9 and pro-MMP-9ΔOG. These data are displayed as histograms inFIGS. 4A-E and are summarized in Table 1, herein below. Most probablevalues are reported, since the presence of two subpopulations may imposea bias on the mean value. Standard deviation is reported betweenbrackets. The values correspond to the wild type pro-MMP-9 (n=90) andpro-MMP-9ΔOG mutant (n=120). Lobe-to-lobe distances were measuredbetween the peaks in the XZ cross-section. Values were extracted only ifthe orientation of the protein on the surface allowed identification oftwo distinct domains (n=83). The separation between lobes inpro-MMP-9ΔOG could not be distinguished.

TABLE 1 Wild type Pro-MMP-9ΔOG Height 34 (7.5)   22 (2.9) Width 190(33)   130 (13) Lobe-to-lobe 78 (9.5) N/A

The most probable height values were 34 Å and 22 Å, for the wild typeand mutant respectively. Comparison to the possible height valuesextracted from the crystal structures [Elkins, P. A., Ho, Y. S., Smith,W. W., Janson, C. A., D'Alessio, K. J., McQueney, M. S., Cummings, M.D., and Romanic, A. M. (2002). Acta Crystallogr D Biol Crystallogr 58,1182-1192; Cha, H., Kopetzki, E., Huber, R., Lanzendorfer, M., andBrandstetter, H. (2002). J Mol Biol 320, 1065-1079] suggest a flatteningof the protein image, which could arise from interactions with thesurface, or some compression of the structure under the AFM tip. Theshape of the histogram of FIG. 4A reveals that the wild type protein isdistributed into two subpopulations, as opposed to the OG-deleted mutant(FIG. 4B), which has a single peak in the distribution. The mostprobable width values were 190 Å and 130 Å for the wild type (FIG. 4C)and pro-MMP-9ΔOG (FIG. 4D), respectively. This difference indicates thatthe OG domain has a significant contribution to the width of the wildtype protein. Some of the spread in height and width values is expectedto occur as a consequence of different binding configurations of theprotein to the surface. Because the protein is not spherical, bindingstates with the major axis oriented at different angles with respect tothe surface normal will lead to different maximum heights and widths asmeasured by AFM. Thus, the height would vary as the cosine of thisangle, and the width as the sine.

The most probable lobe-to-lobe distance values were 78 Å for wild-typepro-MMP-9 (FIG. 4E). This distance compares favorably to lobe-to-lobevalues obtained by SAXS. The range of possible values from the SAXSmodel is in the range of 75-87 Å, depending on the allowed orientationsof the individual domains.

Example 6 Single Molecule Imaging Statistical Analysis Coupled with SAXSReveal Protein Domain Flexibility Mediated by OG Domain

Results

A striking feature contrasting the two enzyme species is that the spreadin both width and height values for the wild type is significantlylarger than for the mutant (FIGS. 4A-E). Such differences could arisefrom the additional degrees of freedom lent to the wild-type structureby the OG domain, as opposed to the mutant, where the two lobes are moreconfined. The heterogeneity of sizes results from two main variables:the different orientations of the protein on the surface and differentprotein conformations. As the mutant contains no OG domain, it has areduced conformational heterogeneity, meaning the spread of values stemsmainly from different orientations on the surface.

The effect of the OG domain on protein flexibility is clearly observedin FIG. 4E which reports measurements of lobe-to-lobe distances. Thespread of distances ranges from 55-85 Å and can be divided to twosubpopulations. Remarkably, these results support the existence ofmultiple enzyme conformations mediated by flexible molecular nature ofthe OG domain. FIG. 4F presents some possible models of various proteinconformations based on the derived lobe-to-lobe distances. Thelobe-to-lobe distances were allowed to vary within one standarddeviation (9.5 Å). The various OG domain conformations presented in FIG.4F were calculated using the structural modeling program RAPPER [38,39].

Protein flexibility of pro-MMP-9, detected by this novel molecularanalysis provides new molecular insights into the overall structure anddynamics of the enzyme, highlighting the structure-function uniquenessof pro-MMP-9 over other members of the MMP family, including gelatinaseA/MMP-2 [21]. For instance, in collagenase-1/MMP-1, the flexibility of amuch shorter linker region is further constrained by interactionsbetween the hemopexin domain and the pro-domain [40]. Similarly, inMMP-2 the second blade of the hemopexin domain is linked to thefibronectin domain through a hydrogen bond (Morgunova et al. Science1999).

Example 7 The Role of Protein Domain Flexibility in Enzymatic Functionof pro-MMP-9

Results

MMP-9 is a secreted enzyme, and it is not clear how it is targeted tothe right location and how its activity is controlled in thepericellular space [41]. Specifically, it is not clear what the roles ofthe various domains are in mediating effective protein-substrate andprotein-protein interactions during catalysis. The full-length structureof pro-MMP-9 reported here introduces novel insights into the structureof this enzyme and to its apparent domain flexibility. In particularly,the reported results raise the possibility that the observed proteinflexibility in MMP-9 is required to mediate its function.

The contribution of the OG domain was postulated in earlier work to be aspacer moiety that allows independent movement of the terminal domains[19, 42, 43]. Interestingly, a bioinformatics “BLAST” search [44, 45] ofall available databases revealed that the OG domain in pro-MMP-9 ishomologous to similar disordered domains in a number of cell surfaceassociated and ECM binding proteins (see Table 2, herein below). Theresults reported in Table 2 lie above the default E-value threshold.Identity and similarity values are in percentage.

TABLE 2 Protein name Organism Identity Similarity Anchor region ofsurface protein Gram-positive 42 62 cocci Outer membrane receptorHaemophilus 43 51 proteins, mostly Fe transport influenzaeHemoglobin-binding protein Haemophilus 42 50 influenzaeCellulose-binding protein B Eubacterium 45 52 cellulosolvens Collagenadhesion protein Bacillus 36 44 thuringiensis serovar israelensis

Remarkably, close structural homology was found between the OG linkerand overall domain organization of pro-MMP-9 and the fungi cellulase[29, 31], for which the role of the linker in cellulase was proposed tomediate protein-cellulose binding and enzyme migration in intactmatrices. This suggests that pro-MMP-9 mediates its biological functionand enzymatic activities by cell surface association and/or interactionswith solid substrates (e.g. ECM). Recently, Owen et al. describedTIMP-1-resistant MMP-9 activity at the cell surface of neutrophils [46].One way to achieve tethering of the MMP-9 hemopexin domain to the cellmight be through interactions of the MT6-MMP/TIMP-1 complex at the cellsurface. The present data suggests that OG domain flexibility allows theN-terminus of MMP-9 to access complex substrate networks (e.g. collagenlike molecules) in the pericellular environment. Stabilization of suchprotein-substrate interactions may be achieved by non specificprotein-protein interactions mediated by the proline-rich sequences [47]residing in MMP-9OG domain. In contrast, the hemopexin-like domain andthe fibronectin domain in pro-MMP-9 were shown to stoichiometricallybind substrates with great affinity [48, 49]. This suggests thatpro-MMP-9 mediates its catalytic activities via both specific andnon-specific interactions with its substrates. In this molecularscenario the two terminal domains will provide substrate specificitywhile the OG flexible domain is used to destabilize, for example, thetertiary structure of collagen type substrates via weak nonspecificinteractions.

Importantly, correct interaction with TIMP-1, LRP-1 and megalin requiresOG domain involvement to achieve proper orientation of thehemopexin-like and catalytic domains [19]. The OG-deleted mutant showeddecreased affinity to these molecules, suggesting the OG-domain isessential for regulating the bioavailability of active MMP-9. Althoughthe single molecule imaging results indicate that the spacing betweenthe terminal domains is not constant, the quasiglobular shape of the OGdomain, as obtained by SAXS model reconstruction, assures minimalseparation between the two domains, allowing binding of regulators tothe C-terminal domain without steric hindrance from the N-terminalcatalytic domain. Such domain flexibility is not observed for MMP-2.This may explain why MMP-9 can bind directly to LRP-1 while MMP-2requires the formation of precursor complex with TIMP-2 [50] or withthrombospondin [51] to achieve effective binding to LRP-1.

In order to test the role of the OG domain in collagenolytic activity ofMMP-9 in-situ inhibition assays were performed. Anti-MMP-9—hinge regionwas used (anti-MMP-9 hr from three different vendors: Sigma, Chemiconand Abcam). This commercially available antibody was raised against apeptide within the OG domain. The substrate used wasfluorescently-labeled type-IV collagen that shows increased fluorescenceupon degradation. FIGS. 7A-B shows that the antibody down regulatescollagenolytic activity, either by hindering the flexibility of the OGdomain or by steric hindrance that disrupt enzyme-substrate contacts asshown by in-situ zymography.

Fluorescently labeled type-IV collagen was overlaid on theMMP-2/9-producing cells HT-1080. Both MMP-2 and MMP-9 that are producedby this cell line are capable of degrading collagen type-IV, however itwas shown that more than 70% of the purified MMPs are MMP-9 that show4-fold specific activity towards collagen type-IV with relation toMMP-2. The collagenolytic activity was examined at the periphery of thecells with (FIG. 7A) or without (FIG. 7B) anti-MMP-9 hr. Upon theaddition of anti-MMP-9 hr the collagenolytic activity is more confinedin comparison to the diffusive activity of the reference.

Taken together, it may be hypothesized that affecting the OG domainreduces the collagenolytic potency whereas the gelatinolytic activity isunaffected.

CONCLUSIONS

This work represents the first experimental structural determination offull-length human pro-MMP-9 revealed by novel combination of structuralanalyses. A combination of single molecule imaging and SAXS was utilizedto derive a comprehensive molecular model providing structural anddynamic insights to this important enzyme. Remarkably, the presentresults demonstrate the presence of a flexible and unstructured OGdomain bridging the catalytic enzyme core and the hemopexin domain. Thisstructure endows pro-MMP-9 with unique domain architecture relative toother family members. Such structural exclusiveness may be utilized forthe design of isoform selective inhibitors for MMP-9. The design ofregulators for MMP-9 may be targeted at restricting its domainflexibility, which may block its pathological activity in specificdisease states.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank

Accession numbers mentioned in this specification are hereinincorporated in their entirety by reference into the specification, tothe same extent as if each individual publication, patent or patentapplication or GenBank Accession number was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

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What is claimed is:
 1. A method of regulating an activity ofmetalloproteinase 9 (MMP-9), the method comprising contacting the MMP-9with an agent which specifically interacts with an OG domain of theMMP-9, thereby regulating the activity of MMP-9.
 2. The method of claim1, wherein said MMP-9 is native MMP-9.
 3. The method of claim 1, whereinsaid activity is a collagenolytic activity.
 4. The method of claim 1,wherein said activity is a gelatinolytic activity.
 5. The method ofclaim 1, wherein the regulating is up-regulating.
 6. The method of claim1, wherein the regulating is down-regulating.
 7. The method of claim 1,wherein said agent comprises a polypeptide agent.
 8. The method of claim1, wherein said polypeptide agent comprises an antibody.
 9. The methodof claim 1, wherein said agent comprises a small molecule.
 10. A methodof identifying an agent capable of specifically regulating MMP-9, themethod comprising determining whether the agent is capable ofinteracting with an OG domain of MMP-9, said agent being a putativeMMP-9 specific regulator.
 11. The method of claim 10, wherein saiddetermining is effected by comparing a structure of the agent to astructure of an OG domain of MMP-9.
 12. The method of claim 10, whereinsaid determining is effected by contacting said agent with an isolatedOG domain of MMP-9.
 13. The method of claim 10, wherein said agentcomprises a polypeptide.
 14. The method of claim 13, wherein saidpolypeptide comprises an antibody.
 15. The method of claim 10, whereinsaid agent comprises a small molecule.
 16. A method of treating a MMP-9mediated medical condition, the method comprising administering to asubject in need thereof a therapeutically effective amount of an agentwhich specifically interacts with an OG domain of MMP-9, therebytreating the MMP-9 mediated medical condition.
 17. The method of claim16, wherein said agent is identified according to claim
 7. 18. Themethod of claim 16, wherein said agent comprises a small molecule or apolypeptide agent.
 19. The method of claim 18, wherein said polypeptideagent comprises an antibody.
 20. A molecule capable of specificallyregulating an activity of MMP-9, wherein the molecule interacts with anOG domain of the MMP-9, with the proviso that the molecule is not anon-humanized antibody.
 21. A humanized antibody comprising an antigenrecognition domain which specifically interact with an OG domain ofMMP-9.
 22. A pharmaceutical composition comprising as an activeingredient the molecule of claim 20 and a pharmaceutically acceptablecarrier.
 23. The pharmaceutical composition of claim 22, wherein saidmolecule comprises a humanized antibody comprising an antigenrecognition domain which specifically interacts with said OG domain ofMMP-9.